Effect of cooling of equine spermatozoa before freezing on post-thaw motility: Preliminary results

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ELSEVIER EFFECT OF COOLING OF EQUINE SPERMATOZOA BEFORE FREEZING ON POST-THAW MOTILITY: PRELIMINARY RESULTS E.C. Crockett, 1 J.K. Graham, 1 J.E. Bruemmer, 2 and E.L. Squires 1 Departments of 1physiology and 2Animal Science Colorado State University, Fort Collins, CO 80523 Received for publication: January l ] , 2000 Accepted: October 31, 2000 ABSTRACT The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5°C and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05)• The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 °C) or after cooling (5 °C), and spermatozoa were frozen after being cooled to 5 °C for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25°C then cooled for 12 h to 5°C had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5°C (13%). In the third experiment, spermatozoa were centrifuged f~r seminal plasma removal, resuspended at spermatozoal concentrations of 50, 250 or 500 x 10/r~., cooled to 5°C for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10/mL had higher (P < 0.05~percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10 spermatozoa/mL (17%). We recommend that semen be centrifuged at 25°C to remove seminal plasma, suspended to 250 x 106 spermatozoa/ml and held at 5°C for 12 h prior to freezing. © 2001 by ElsevverSctence Inc

Key wo"ds: stallion, semen, cooling, centrifugation, cryopreservation

Acknowledgments This study was supported by benefactors of the Preservation of Equine Genetics Program, the Colorado Racing Council and the Lucy Whittier Foundation. Thenogenology 55:793-803, 2001 © 2001 Elsevier Sctence Inc.

O093-691X/O1/$-see front matter PII: S0093-691X(01)00444-7

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INTRODUCTION Use of cooled and frozen stallion semen is gaining wider acceptance in the equine industry. The procedures used to cool semen are relatively simple and stallion spermatozoa can be cooled properly at most breeding farms. However, cryopreservation of semen requires sophisticated equipment and expertise beyond the capabilities of many breeding farms. Several procedures have been developed that successfully cryopreserve stallion spermatozoa, but the procedures use freshly collected semen and require that the stallion be located at the facility where cryopreservation takes place (1,3,9,12,17). The ability to collect and ship cooled stallion semen from one location to a specialized facility for subsequent freezing would be advantageous because the stallion could remain at home while his semen is frozen by specialists elsewhere. Several investigators reported that removing seminal plasma from stallion spermatozoa before cooling and storing at 5 °C is beneficial (4,7,14). However, completely removing the seminal plasma from the spermatozoa before cooling had detrimental effects. Leaving 5 to 20% seminal plasma in the semen sample maintained the highest spermatozoal motility (7,14) and acrosomal mtegrity (4). To cryopreserve stallion spermatozoa at a location distant from the stallion it is necessary to freeze cooled, shipped spermatozoa. No reports exist stating whether spermatozoa should be diluted, shipped and subsequently prepared for cryopreservation at the distant facility or if semen should be centrifuged first and then shipped. In addition, the length of time that equine spermatozoa can be stored at 5°C before freezing while still retaining acceptable post-thaw motility has yet to be determined. Reliable methods for cooling and cryopreserving stallion semen have not been investigated. Although 50 x 106/mE is the recommended concentration for cooling spermatozoa, this concentration is too low for cryopreservation, since a great number of straws would be needed for each insemination. Heitland et al. (5) determined that spermatozoa frozen in a skim milk-egg yolk extender at concentrations of 20, 200 and 400 x 106/mE had higher percentages of total motile (51, 51 and 50%, respectively) and progressively motile (41, 44 and 43%, respectively) spermatozoa compared to spermatozoa frozen at 800 or 1,500 x 106/mE (41 and 32; 35 and 27%, respectively). The overall objectives of this study were to determine the most appropriate extender, package, time of centrifugation for seminal plasma removal, spermatozoal concentration and length of time after collection that spermatozoa can be cooled prior to cryopreservation on quality of cryopreserved semen. MATERIALS AND METHODS Experiment 1 A 3 x 2 x 2 factorial experiment was designed to determine which of three cryopreservation extenders and which of two spermatozoal package volumes (0.5- or 2.5-mL) was most effective in preserving stallion spermatozoa held at 5°C for either 2.5 or 24 h before freezing. Single ejaculates from each of eight stallions were collected using an artificial vagina (AV) and the semen was diluted 1:1 (v:v) in a HEPES-buffered extender (HBM; 277.5 mM glucose; 8.3 mM

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lactose; 5.0 mM raffinose; 1.7 mM sodium citrate dihydrate; 2.5 mM potassium citrate; 29.8 mM HEPES; adjusted to pH 7.2) at 37°C, within 5 min of coUection. The semen was then immediately washed by centrifugation at 300 x g for 8 min, the supernatant was discarded and the spermatozoal pellets were resuspended in 1 mL of HBM. All spermatozoa from an individual stallion then were pooled, the spermatozoal concentration was determined by6 spectrophotometric assay and the spermatozoal samples were diluted to 200 to 300 x 10 spermatozoa/mL in either a skim milk-egg yolk-sugar extender (SMEY; 154.8 mM glucose, 4.2 mM lactose, 0.5 mM raffinose, 0.85 mM sodium citrate dihydrate, 1.25 mM potassium citrate, 29.8 mM HEPES, 51.5 mg/mL skim milk powder, I mg/mL ticarcillin, 2% egg yolk by volume, adjusted to a pH of 7.2), skim milk-egg yolk-salt extender (EYCO; 18.5 mM NaC1, 5 mM KCL, 0.6 mM KH2PO4, 17.8 mM NaHCO3, 1.2 mM MgSO4, 0.8 mM CaC12, 42 mM fructose, 18.5 mM glucose, 0.6 mM Na-pyruvate, 6.5 mM Na-lactate, 5 mM HEPES, 1.5 mg/mL BSA ' 51.5 mg/mL skim milk powder, 1 mg/mL ticarcillin, 2% egg yolk by volume, pH adjusted a to 7.2) or skim milk (SM, pH 6.8-7.0). Samples were coded and a visual estimation of the percentage of progressively motile spermatozoa (PM) in each sample was conducted by a single observer after samples were diluted 1:9 (volume of sample : volume of respective extender). For each stallion, two 2.5-mL aliquots of spermatozoa in each extender were placed in a 100-mL water bath at 25°C which was placed in 5°C air and cooled to 5°C over 2.5 h. One aliquot was cryopreserved 2.5 h after the onset of cooling while the second aliquot was maintained at 5 °C for 24 h before cryopreservation. After cooling for 2.5 or 24 h, samples were diluted with an equal volume of respective extender (SMEY, EYCO, or SM) each containing 10% glycerol by volume. The samples were mixe~by slowly inverting them three times. The spermatozoa were packaged into one 2.5-mL straw (a 5-mL straw cut in half) and two 0.5-mL polyvinylchloride straws ~ and then frozen in static nitrogen vapor 3 cm above liquid nitrogen for 10 min before plunging the straws into liquid nitrogen for storage at - 196 °C. The 2.5-mL straws were thawed in a water bath at 50°C for 45 sec and then placed in a 37°C water bath for 30 seconds. The 0.5-mL straws were thawed in a 37°C water bath for 30 seconds. Spermatozoa from b o ~ 0.5-mL straws were pooled before analysis using a computer-automated sperm analysis system (CASA). Analyses were conducted on a single_6-pL aliquot of spermatozoa for each sample placed on preheated (37 o C) CELL-VU T u I~ slides for analysis. System parameter settings for these analyses were 15 frames acquired at 30 frames per second; magnification factor 1.95; minimum contrast 75, minimum size 4 pixels; lower and upper static a

EZ-Mixin BF, Animal Reproduction Systems, Chino, CA. b c d

Minitub, Verona, WI. IMV International Corporation, Minneapolisl MN. Hamilton Thorne HT-IVOS Motility Analyzer, Hamilton Thome Research, Danvers, MA.

e Fertility Technologies, Inc., Natick, MA.

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size limits of 0.7 and 7.75, respectively; lower and upper static head intensities of 0.43 and 1.99, respectively; minimum velocity of the average path of motile spermatozoa (VAP) was 20 lam/second. A minimum of four random areas on each slide and a minimum of 100 spermatozoa were analyzed. Experiment 2 Results from the first experiment indicated that spermatozoa could not be held at 5°C for 24 prior to freezing. Therefore, the objectives of Experiment 2 were to determine if spermatozoa could tolerate cooling for 12 h before freezing and if spermatozoa should be centrifuged before or after cooling to 5°C. Single ejaculates from ten light-horse stallions were collected before the study using an AV to obtain seminal plasma. Seminal plasma was collected by placing each ejaculate into several 50-mL conical tubes and centrifuged at 400 x g for 12 minutes. The supernatant was collected and centrifuged a second time at 650 x g for 20 min to remove any remaining spermatozoa. The seminal plasma was then frozen in 1-mL aliquots at -20°C until needed, when it was thawed in a water bath at 25°C. 9 Single ejaculates from the same stallions containing at least 4.2 x 10 spermatozoa and 50% PM were diluted in §M extender without glycerol and split into six aliquots of 10 mL, each containing 700 x 10 spermatozoa. Three aliquots from each ejaculate were centrifuged at 275 x g in 15-mL conical tubes for 7.5 minutes. After centrifugation, the supernatant from each sample was removed and the spermatozoal pellet was resuspended with SM containing ticarcillin (lmg/mL). Then 1 mL of each sample was removed and 1 mL of the stallion's own seminal plasma (approximately 10% of total volume) was added and the samples were cooled to 5 °C. One of these aliquots was held at 5°C for 2 h, one was held at 5°C for 6 h and one was held at 5°C for 12 h before freezing. The remaining three uncentrifuged aliquots were cooled to 5°C for a period of 2, 6 or 12 h at which time they were centrifuged at 275 x g for 7.5 min at 5°C and processed as described above. Samples to be incubated 2 h were cooled to 5°C by placing the 15-mL conical tubes in a 100-mL water bath at 25°C and placing the water bath in 5°C air for 2 hours. Samples to be incubated for ~ and 12 h were packaged in Whirlpack bags and placed in a passive cooling device (EquitainerrM-). Spermatozoa were cryopreserved by diluting each aliquot (10 mL) with an additional 10 mL of SM extender containmg ticarcillin (1 mg/mL), 8% glycerol (v:v) and 8% egg yolk (v:v), yielding final glycerol an~ egg yolk concentrations of 4% and a final spermatozoal concentration of approximately 25 x 10 spermatozoa/mE The spermatozoa were then packaged into 0.5-mL polyvinylchloride straws g and frozen by suspending the straws in static nitrogen vapor 3 cm above liquid nitrogen (-165 °C _+5 °C) before plunging into liquid nitrogen, then storing in liquid nitrogen (-196°C). A single straw from each treatment was thawed in a 37°C water bath for 30 seconds and analyzed using CASA, as described above.

f Equitainer I TM,Hamilton Thome Research, Danvers, MA. g IMV International Corporation, Minneapolis, MN.

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Experiment 2 The motion characterisUcs for spermatozoa centrifuged at either 25 °C or 5 °C are presented (Table 2). Samples centrifuged at 25°C had higher (P < 0.05) TM after cenmfugation than did samples centrifuged at 5 °C. Similar post-thaw TM were obtained for both centrifugation treatments. However, the PM of samples were higher (P < 0.05) post-centrifugation and postthaw when spermatozoa were centrifuged at 25°C compared to 5°C. The VAP of spermatozoa were similar (P > 0.05) regardless of whether spermatozoa were centrifuged at 25 or 5°C.

Table 2. The percentage Of total motile (TM), progressively motile (PM) and the average path velocity (VAP, pm/sec) for equine spermatozoa post-centrifugation and post-thaw when centrifuged at 25°C or 5°C and frozen Post-centrifugation

Post-thaw

Centrifugation temperature

TM (%)

PM (%)

VAP (pm/sec)

TM (%)

PM (%)

VAP (prn/sec)

25 ° 5°

75~ 62 b

60~ 48 b

87 a 84 a

48 a 34 a

30~ 19D

86 a 73 a

SEM

2

2

5

3

2

5

a,b Values within columns with different superscripts differ (P < 0.05).

An interaction between centrifugation treatment and cooling time was observed (Table 3 and Table 4). The TM of spermatozoa were similar when samples were centrifuged before cooling for 2, 6 or 12 h (Table 3). Centrifugation of spermatozoa after having been cooled for 12 h resulted in lower (P < 0.05) (48%) TM than for spermatozoa having been cooled for only 2 or 6 h (73 and 65%, respectively) before centrifugation (Table 3). Samples stored for 6 h at 5 °C before centrifugation had lower TM (65%) than those stored for 2 h (73%) before centrifugation. The PM was similar (P > 0.05) for spermatozoa centrifuged before cooling and then incubated at 5 °C for 2, 6 or 12 h before freezing (Table 3). When spermatozoa were cooled for 12 h and then centrifuged a lower PM (35%) was observed than for samples centrifuged after incubation at 5 °C for 2 h (60%; Table 3). Spermatozoa centrifuged before coohng for 12 h had a higher (P < 0.05) PM than samples centrifuged after cooling for 12 h (Table 3). The VAP for all samples were similar (P > 0.05;Table 3). Post-thaw motility parameters for equine spermatozoa cooled for 2, 6, or 12 h before freezing are presented (Table 4). The post-thaw TM of spermatozoa were similar within centrifugation treatment regardless of cooling for 2, 6 or 12 h at 5 °C (Table 4). Centrifugation of spermatozoa after cooling to 5°C for 12 h resulted in lower (35%) TM (P < 0.05) than for spermatozoa that were centrifuged before cooling to 5°C for 12 h (58%; Table 4). The post-thaw PM were similar for samples centrifuged before cooling (Table 4). When spermatozoa were cooled for 2 or 6 h and then centrifuged PM, were similar (P > 0.05, Table 4). Spermatozoa cooled for 6 or 12 h

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6 10 spermatozoa/mL before freezing (~able 5). Post-thaw percentages of viable spermatozoa of samples cooled at 50, 250 or 500 x 10- spermatozoa/mL before freezing were different (P < 0.05, Table 5).

Table 5. Percentages of post-thaw total motile (TM), progressively motile (PM), viable equine spermatozoa cooled at varied concentrations for 12 h at 5°C and then frozen Treatment 50 x 106~mL 250 x 10-/mL 500 x 10ta/mL SEM a,b,c

TM (%) 38 abe 42 b 32 c 2

PM (%)

Viable spermatozoa (%)

25 b 23 a 17c

60 a 53 b 45 c

2

2

Values within columns with different superscripts differ (P < 0.05).

DISCUSSION Several different freezing regimens have been used to cryopreserve stallion spermatozoa (3,9,12,17). However, because diluents and procedures used in each regimen are different direct comparison of the results for these techniques are inappropriate, and controlled studies comparing the different procedures have not been reported. Previous studies reported using a SM based extender for freezing equine spermatozoa (9,10, 11,12). Skim milk extenders generally require relatively slow cooling of spermatozoa to 5 °C over a 2- to 2.5-h time period before freezing. Incubating stallion spermatozoa at 5°C for 24 h before cryopreservation resulted in fewer percentages of post-thaw PM spermatozoa. However, removing seminal plasma at 25 °C and incubating spermatozoa at 5 °C for 6 or 12 h before cryopreservation resulted in percentages of PM after freezing and thawing that were similar to those for incubating spermatozoa for 2 to 2.5 hours. This would allow semen to be collected at one location and shipped to a facility having expertise in freezing of equine spermatozoa. Spermatozoa survived cryopreservation equally well when packaged into either 0.5- or 2.5mL straws. Samper (15) reported similar percentages of motile spermatozoa when stallion spermatozoa were frozen in 0.5-, 2.5- and 5-mL straws (40, 38, and 42% respectively). Packaging spermatozoa in 0.5-mL straws was hypothesized to provide a more uniform freeze, because of the higher surface to volume ratio of this packaging system, and result in a higher recovery of motility after freezing and thawing. Although the freeze rates of the diluents within individual straws at different distances from the straw surface were not determined in this experiment, any such differences did not affect the overall percentages of motile spermatozoa after freezing and thawing. When freezing equine spermatozoa, seminal plasma is removed either by collecting only the spermatozoa-rich fraction of the ejaculate or by centrifugation (9,18). Centrifuging spermatozoa

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to remove seminal plasma can be detrimental to spermatozoal survival, but can be minimized by using low centrifuge forces (13). In our study we did not observe major detrimental effects of centrifugation unless centrifugation was conducted at 5°C after spermatozoa had been incubated for 6 or 12 hours. Therefore, if spermatozoa are to be stored for more than 2 to 2.5 h before freezing the spermatozoa must be centrifuged to remove the seminal plasma at 25 °C. In Experiment 1, we determined that egg yolk helped maintain sperm motility after freezing and thawing. Bedford et al. (2) demonstrated that if semen was centrifuged to remove seminal plasma and then cooled for 24 h at 5°C, egg yolk in the extender was beneficial (51%) to spermatozoa, compared to an extender containing only skim milk (42%, P < 0.05). Jasko et al. (6) also reported that egg yolk positively influenced spermatozoa cooled to 5 °C and held for 24 hours. When spermatozoa were centrifuged to remove the seminal plasma and resuspended in an extender containing egg yolk and cooled to 5 °C, the percentage of progressively motile spermatozoa was higher (38%) when compared to spermatozoa resuspended m skim milk alone (22%, P < 0.05). The improved survival of spermatozoa frozen in SMEY and EYCO compared to SM may also be attributed to the sugars, salts and Hepes contained in these extenders compared to only skim milk and glucose in the SM extender. 6Leipold et al. (8) reported that spermatozoa frozen at concentrations of 400 and 1600 x 1 0 / m L had similar percentages6of TM and PM. In the present study, spermatozoa frozen at lower concentration of 250 x 10-/mL, maintained a higher percentage ~ total motile and progressively motile spermatozoa than spermatozoa cooled at 500 x 10- spermatozoa/mL before cryopreservation. In summary, these results indicate that stallion spermatozoa ca~ be centrifuged to remove the seminal plasma at 25 °C, the spermatozoa resuspended to 250 x 10- spermatozoa/mL, cooled to 5°C and then held at 5°C for 12 h before freezing without affecting the percentages of motile spermatozoa in the samples after freezing and thawing. This procedure would permit the semen from at least some stallions to be collected, centrifuged on the farm and shipped to a facility specializing in cryopreserving of equine spermatozoa. However, the spermatozoa must reach the facility within 12 h of collection. Additional experiments are needed in which multiple straws from an ejaculate are evaluated, as well as trials to determine the fertilizing potential of spermatozoa held for 12 h before freezing. REFERENCES 1. Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci 1987;7:145-173. 2. Bedford S J, Graham JK, Amann RP, Squires EL, Pickett BW. Use of two freezing extenders to cool stallion spermatozoa to 5 °C with and without seminal plasma. Tberiogenology 1995;43:939-953. 3. Cristanelli MJ, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed, frozen and thawed by a new procedure. Theriogenology 1984;22:39-45 4. Dawson GR, Webb GW, Pruitt JA, Loughlin TM, Ares MJ. Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa. Proc ENPS, 1999; 77-82.

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5. Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C. Factors affecting motion characteristics of frozen-thawed stallion spermatozoa. Equine Vet J 1996;28(1):4753. 6. Jasko DJ, Hathaway JA, Schaltenbrand VL, Simper WD, Squires EL. Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 1992;37:1241-1252. 7. Jasko DJ, Moran DM, Farlin ME, Squires EL. Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Theriogenology 1991 ;35:1059-1067. 8. Leipold SD, Graham JK, Sqmres EL, McCue PM, Brinsko SP, Vanderwall DK. Effect of spermatozoal concentration and number on fertility of frozen equine semen. Theriogenology 1998;49:1537-1543. 9. Martin JC, Klug E, Gunzel AR. Centrifugation of stallion semen and its storage in large volume straws. J ReprodFertil 1979;27(Suppl):47-51. 10. Nishikawa Y. Studies on the preservation of raw and frozen horse semen. J Reprod Fertil 1975;23(Suppl):99-104. 11. Nishikawa Y, Shinomiya S. Studies on the protective effects of egg yolk and glycerol on the freezability of horse spermatozoa. Proc 7th Intl Congr Anim Reprod AI 1972;2:15451549. 12. Palmer E. Factors affecting stallion semen survival and fertility. Proc 10th Congr Anita AI 1984;3:377. 13. Pickett BW, Sullivan JJ, Byers WW, Pace MM, Remmenga EE. Effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa. Fertil Steril 1975;26:167-174. 14. Pruitt JA, Arns MJ, Pool KC. Seminal plasma influences recovery of equine spermatozoa following in vitro culture (37 °C) and cold-storage (5 °C). Theriogenology 1993;39:291 abstr. 15. Samper JC. Stallion semen cryopreservation: Male factors affecting pregnancy rates. Proc Soc Study Theriogenology 1995; 160-165. 16. SAS Institute Inc., SAS User's Guide: Statistics, Cary, NC: Statistical Analysis Systems Institute Inc. 1985. 17. Tischner M. Evaluation of deep-frozen semen in stallions. J Reprod Fertil 1979;27(Suppl):53-59. 18. Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM. Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. Theriogenology 1987;28(5):709-718.